Iron is critical for growth of bacterial pathogens due to the need for iron in heme and iron-sulfur (Fe-S) clusters. To combat infection, the human host uses two general strategies that exploit this iron dependence: iron sequestration to minimize bioavailable iron for the pathogen and attack of the pathogen's iron metalloproteins via generation of oxidative stress. Our long-term goal is to characterize the genetic and biochemical systems utilized by bacterial pathogens to preserve intracellular iron homeostasis during stress. The objective of this proposal is to determine the biochemical mechanisms used by the Suf pathway to build Fe-S clusters during iron starvation and oxidative stress. The sufABCDSE operon is activated in bacteria to build essential Fe-S clusters during exposure to oxidative stress and iron starvation. The Suf pathway is conserved in many bacterial pathogens such as Shigella and Mycobacterium tuberculosis. Shigella is responsible for the deaths of 11 million people each year due to bacillary dysentery, the majority of which are children under the age of five. The suf operon may be important for Shigella pathogenesis since it is transcribed as Shigella enters the intracellular stage of its pathogenic lifecycle. M. tuberculosis is the causative agent of tuberculosis and directly causes 2 million deaths each year. In M. tuberculosis, the suf genes are essential since deletion of the suf genes is lethal in M. tuberculosis and related Mycobacteria. Despite its importance, the molecular details of in vivo Suf function are still unclear. The SufS enzyme is a cysteine desulfurase that provides sulfur for Fe-S cluster assembly, while the functions of SufA, SufB, SufC, SufD, and SufE are not fully known. Our Aims are to (1) characterize the step-by-step path of sulfur transfer from SufS to its ultimate destination for Fe-S cluster assembly;(2) identify the site(s) of Fe-S cluster assembly in the Suf operon;and (3) determine the function of SufC ATPase activity in the multi-protein SufBCD complex during Fe-S cluster assembly. We will use methods in protein chemistry, bioinorganic chemistry, molecular biology, and microbial genetics to accomplish our Aims in the facile model organism Escherichia coli. Lay Statement: Disruption of bacterial iron homeostasis is a key mechanism used by mammalian cells to limit bacterial growth during infection because iron is an essential nutrient for many pathogenic bacteria. We believe that targeting the Suf Fe-S cluster biogenesis pathway with novel antibiotics could be a strategy for assisting the host defenses in disrupting bacterial iron homeostasis. Our proposal is designed to characterize the Suf pathway so that we might design specific inhibitors against the Suf proteins that will act as a new class of antibiotics